Assessment of PAX6 alleles in 66 families with aniridia.

We report on PAX6 alleles associated with a clinical diagnosis of classical aniridia in 81 affected individuals representing 66 families. Allelic variants expected to affect PAX6 function were identified in 61 families (76 individuals). Ten cases of sporadic aniridia (10 families) had complete (8 cases) or partial (2 cases) deletion of the PAX6 gene. Sequence changes that introduced a premature termination codon into the open reading frame of PAX6 occurred in 47 families (62 individuals). Three individuals with sporadic aniridia (three families) had sequence changes (one deletion, two run-on mutations) expected to result in a C-terminal extension. An intronic deletion of unknown functional significance was detected in one case of sporadic aniridia (one family), but not in unaffected relatives. Within these 61 families, single nucleotide substitutions accounted for 30/61 (49%), indels for 23/61 (38%), and complete deletion of the PAX6 locus for 8/61 (13%). In five cases of sporadic aniridia (five families), no disease-causing mutation in the coding region was detected. In total, 23 unique variants were identified that have not been reported in the Leiden Open Variation Database (LOVD) database. Within the group assessed, 92% had sequence changes expected to reduce PAX6 function, confirming the primacy of PAX6 haploinsufficiency as causal for aniridia.

Targeting the tetraspanin CD81 blocks monocyte transmigration and ameliorates EAE.

Leukocyte infiltration is a key step in the development of demyelinating lesions in multiple sclerosis (MS), and molecules mediating leukocyte-endothelial interactions represent prime candidates for the development of therapeutic strategies. Here we studied the effects of blocking the integrin-associated tetraspanin CD81 in in vitro and in vivo models for MS. In an in vitro setting mAb against CD81 significantly reduced monocyte transmigration across brain endothelial cell monolayers, both in rodent and human models. Interestingly, leukocyte as well as endothelial CD81 was involved in this inhibitory effect. To assess their therapeutic potential, CD81 mAb were administered to mice suffering from experimental autoimmune encephalomyelitis (EAE). We found that Eat2, but not 2F7 mAb directed against mouse CD81 significantly reduced the development of neurological symptoms of EAE when using a preventive approach. Concomitantly, Eat2 treated animals showed reduced inflammation in the spinal cord. We conclude that CD81 represents a potential therapeutic target to interfere with leukocyte infiltration and ameliorate inflammatory neurological damage in MS.

Intraspinal administration of an antibody against CD81 enhances functional recovery and tissue sparing after experimental spinal cord injury.

We previously demonstrated that the tetraspanin protein CD81 is up-regulated by astrocytes and microglia after traumatic spinal cord injury in rats and that CD81 is involved in adhesion and proliferation of cultured astrocytes and microglia. Since these reactive glial cells contribute to secondary damage and glial scar formation, we studied the effect of local administration of an anti-CD81 antibody in experimental spinal cord injury. Adult rats were subjected to a moderate spinal cord contusion injury and treated for 2 weeks with different doses of the anti-CD81 antibody AMP1 (0.5-5 microg/h) or non-immune IgG (5.0 microg/h). A technique was developed to infuse the antibodies directly into the lesion site via an intraspinal cannula connected to a pump. Functional recovery was monitored during 8 postoperative weeks by means of the Basso, Beattie and Bresnahan (BBB) locomotor rating scale, the BBB subscore and Grid-walk test. At the end of the study, quantitative histology was performed to assess tissue sparing. Our data showed that by itself cannulation of the lesion site resulted in minimal functional and histological impairments. Application of 0.5 microg/h AMP1 resulted in a marked functional recovery (BBB 2 points; Grid-walk 30% less errors compared to control). This recovery was accompanied by an 18% increase in tissue sparing at the lesion epicentre. No gross histological changes in glial scarring were apparent. Our data demonstrate beneficial effects of an anti-CD81 antibody on functional recovery in spinal cord injured rats and suggest that this effect is mediated through a reduction in secondary tissue loss.

A horseradish peroxidase-light and electron microscopic study of immunoliposomes utilized for intracellular delivery to the rat striatum.

Liposomes can deliver plasmid DNA, viruses, antisense oligonucleotides, and pharmacological agents to the central nervous system. Conjugation of antibodies to liposomes increases delivery specificity. Immunoliposomes created with Thy 1.1 antibody have previously been shown to be effective for neuronal delivery. The intracellular delivery of these immunoliposomes is evaluated by light and electron microscopy. Thy 1.1 conjugated liposomes were loaded with horseradish peroxidase and stereotactically injected into rat striatum. On light microscopy, immunoliposomes were concentrated within 0.2 mm of the injection site 8 h following delivery but, 24 h post-operatively, had diffused more than 0.5 mm from the injection site. With transmission electron microscopy, immunoliposomes were observed entering numerous neurons and some astrocytes in a process distinct from the clathrin-coated pit mechanism. These findings suggest that Thy 1.1 immunoliposomes are effective for intracellular delivery in vivo and their endocytosis occurs independently of a coated pit process. The research has helped to elucidate alternative mechanisms for immunoliposomal delivery. A more fundamental understanding of these attributes is needed to achieve the therapeutic potential of immunoliposomes.

CD81 and microglial activation in vitro: proliferation, phagocytosis and nitric oxide production.

CD81 (TAPA), a member of the tetraspanin family of proteins, is upregulated by astrocytes and microglia after traumatic injury to the rat central nervous system (CNS). To further understand the role of CD81 in the microglial response to injury, we analysed the functional effects of a CD81 antibody, AMP1, on cultured rat microglia. We found that AMP1 suppressed microglial proliferation in a dose-dependent manner. Furthermore, AMP1 stimulated myelin phagocytosis, probably by opsonizing the myelin. The phagocytosis of latex beads, as well as the production of nitric oxide, were not significantly influenced by AMP1. These data indicate that CD81 is involved in an important subset of microglial effector functions after CNS injury.

N-cadherin at the glial scar in the rat.

Following injury to the central nervous system (CNS), astrocytes become reactive and in many cases form a glial scar. Very little is known about the adhesive interactions between astrocytes at the glial scar, even though reactive gliosis and scar formation are a central issue in CNS wound healing. In the present study, we examine the role of cadherin in the process of scar formation using immunohistochemistry and immunoblot methods. When a stab wound was made in the cerebral cortex of the rat, cadherins were consistently upregulated by the reactive astrocytes at the glial scar. Our immunoblot analysis demonstrates that the increase in cadherin immunoreactivity was due to a threefold upregulation of a single protein with a molecular weight of 135 kDa. The size (135 kDa) and location of the immunoreactive protein at regions of cell-cell contact in cultured astrocytes indicates that the immunoreactive protein is N-cadherin. These data are the first to demonstrate that N-cadherin plays a prominent role in the response of astrocytes to injury, including the formation and maintenance of the glial scar.

The expression of TAPA (CD81) correlates with the reactive response of astrocytes in the developing rat CNS.

During the development of the brain, astrocytes acquire the ability to become reactive and form a scar. This change in the astrocytes occurs at approximately the same time that there is a decrease in the regenerative capacity of the CNS. Previous work from our laboratory had revealed that TAPA (Target of Anti-Proliferative Antibody, also known as CD81) is associated with reactive gliosis and the glial scar. TAPA is a member of the tetraspan family of proteins that appears to be associated with the regulation of cellular behavior. In order to define the role of TAPA in relation to the developmentally regulated CNS response to injury, we examined the levels of TAPA and GFAP immunoreactivity in rat pups that received a penetrating cerebral cortical injury. All of the animals injured at postnatal day 9 (PND 9), PND 18, or as adults, exhibited reactive gliosis scar formation when they were sacrificed 10 days after the cortical injury. Of the nine animals injured at PND 2, only three displayed reactive gliosis and scar formation. The remaining six rat pups had either a modest gliotic response or no detectable gliosis. The level of TAPA at the site of injury mimicked the reactive gliosis as defined by GFAP immunoreactivity. In all of the rats with a glial scar, there was a dramatic upregulation of TAPA that is spatially restricted to the reactive astrocytes. These results suggest that the upregulation of TAPA is an integral component of glial scar formation.

Expression of rat target of the antiproliferative antibody (TAPA) in the developing brain.

The present study defines the expression pattern of TAPA (target of the antiproliferative antibody, also known as CD81) in the developing rat brain. TAPA is a member of the tetramembrane spanning family of proteins, and like other members of this family it appears to be associated with the stabilization of cellular contacts (Geisert et al. [1996] J. Neurosci. 16:5478-5487). On immunoblots of the brain, TAPA is present in higher levels than any other tissue examined: muscle, tendon, peripheral nerve, cartilage, liver, kidney, skin, and testicle. Immunohistochemical methods were used to define the distribution of TAPA in the brain. This protein is expressed by ependyma, choroid plexus, astrocytes, and oligodendrocytes. TAPA is dramatically upregulated during early postnatal development, at the time of glial birth and maturation. At embryonic day 18, the levels of TAPA are low, with most of the immunoreaction product being associated with the ependyma, choroid plexus, and the glia limitans. As development continues, the amount of TAPA expressed in the brain increases, and at postnatal day 14 the levels approach those of the adult. This increase in the levels of TAPA at postnatal day 14 is due to upregulation in the gray matter and white matter. Thus, TAPA is found in all glial cells, and the level of this protein correlates with their maturation.

The target of the antiproliferative antibody (TAPA) in the normal and injured rat retina.

PURPOSE: The target of the antiproliferative antibody (TAPA, CD81) is a member of the tetramembrane spanning superfamily of proteins and appears to be involved in the regulation of mitotic activity and the stabilization of cellular contacts [J Neurosci 1996; 16:5478-5487]. The present study examines the distribution of this protein in the normal rat retina and its role in reactive gliosis occurring after retinal injury. METHODS: An immunoblot was used to define the relative level of TAPA in the normal rat retina. The distribution of the protein was examined using indirect immunohistochemical methods. Both of these methods were used to define the upregulation of TAPA in the rat retina injured with a needle scrape. RESULTS: The immunohistochemical analysis of the retina shows that TAPA is found in all layers of the normal retina with a distinct lack of labeling in the inner and outer segments of the photoreceptors. After retinal injury, a dramatic upregulation of TAPA was observed. The immunohistochemistry also revealed a pattern of expression similar to that observed in the normal retina with two notable exceptions: (1) small finger-like projections extending down into the outer segments are immunopositive, and (2) the elevated levels of TAPA can be seen outlining cell bodies in the outer nuclear layer and the ganglion cell layer. CONCLUSIONS: TAPA is found in the normal rat retina and there is a dramatic upregulation of this protein following injury. The distribution of the protein within the retina is consistent with its expression in retinal glia, the Muller cells which span the thickness of the retina, and astrocytes found in the ganglion cell layer. These data suggest that TAPA may play a role in the proliferative response of non-neuronal cells that occurs following a mechanical injury to the retina.

A novel approach to identify proteins associated with the inhibition of neurite growth.

In the present study, a novel combination of techniques was used to identify the genes that may be involved in the lack of axonal regeneration in the mammalian adult central nervous system (CNS). The key features of this approach are: (1) a functional assay that can be affected by antibody perturbation; (2) increased specificity of the polyclonal antiserum by adsorption; (3) the expression cloning of the genes from a lambdagt11 library; (4) amplification of the insert cDNA by PCR; and (5) the direct cycle sequencing of PCR products. In this culture assay system, neurons were plated directly on sections of the rat CNS. This assay system could be used to demonstrate the lack of neuronal attachment to or neurite extension over myelinated regions of the CNS (white matter). This prohibitive nature of the CNS sections could be masked by a rabbit polyclonal antiserum directed against rat CNS white matter. This data indicates that the anti-white matter antiserum recognizes and neutralizes inhibitory molecules on the surface of the sections. Making the assumption that the prohibitive antigen is associated with the cell membrane, the antiserum was adsorbed against a soluble protein fraction of the adult rat brain. This adsorption significantly increased the specificity of the antiserum as demonstrated by immunoblot methods. The adsorbed antiserum was then used to screen the cDNA library of the adult rat brain. The present report describes this novel combination of techniques allowing one to go from a functional tissue culture assay system to defining the molecular basis for the cellular interactions.

Astrocyte growth, reactivity, and the target of the antiproliferative antibody, TAPA.

Reactive astrocytes form a scar after injury to the CNS that many investigators believe contributes to the lack of functional regeneration. In the present study, we identify an astrocytic membrane protein that appears to play an important role in reactive gliosis and scar formation. Cultures of rat astrocytes were used as a model system to produce and to screen monoclonal antibodies that would alter cell growth. One antibody, AMP1, was identified that depresses the mitotic activity of cultured glial cells and alters their morphology. Expression cloning reveals that the antigen on the external surface of the cultured glial cells has a high degree of homology with the human lymphocyte protein called Target of the Anti-Proliferative Antibody (TAPA-1; this rat protein will be referred to as rTAPA). rTAPA is a member of the tetramembrane-spanning superfamily of proteins and, as with other members of this family of proteins, rTAPA is associated with the regulation of cellular interactions and mitotic activity. After an injury to the cerebral cortex, there is a dramatic increase in AMP1 immunoreactivity that is spatially restricted to the reactive astrocytes at the glial scar. This change represents an upregulation of a membrane protein, rTAPA, that is approximately equal to the increase observed for glial fibrillary acidic protein. The high levels of rTAPA at the site of CNS injury and the AMP1 antibody perturbation studies indicate that rTAPA may play a prominent role in the response of astrocytes to injury and in glial scar formation.

Up-regulation of a keratan sulfate proteoglycan following cortical injury in neonatal rats.

The up-regulation of the keratan sulfate proteoglycan (ABAKAN) was examined using indirect immunohistochemical methods. Previous studies indicate that the keratan sulfate proteoglycan is associated with astrocytes in the optic nerve and in the developing rat brain. In model culture systems, this proteoglycan is capable of inhibiting the growth of neurites over laminin. To determine whether the proteoglycan is up-regulated specifically during reactive gliosis, stab wounds were made in the cerebral cortex of early postnatal rats, and the up-regulation of the proteoglycan was related to the developmentally regulated gliotic response to injury. Following a stab wound in the cortex of the late postnatal rat, reactive gliosis was consistently observed along with an up-regulation of ABAKAN. When the cortex was injured on postnatal day 2, there was a variable gliotic response and considerable variation in the regulation of proteoglycan expression. Biochemical analysis revealed that ABAKAN is a large proteoglycan with multiple keratan sulfate side-chains, at least one chondroitin sulfate side-chain and at least one additional carbohydrate chain with a terminal 3-sulfoglucuronic acid. Taken together, these data demonstrate that the boundary proteoglycan ABAKAN is also associated with reactive gliosis during early postnatal development.

A keratan sulfate proteoglycan marks the boundaries in the cortical barrel fields of the adult rat.

The present study uses the monoclonal antibody TED 15 to examine the distribution of a keratan sulfate proteoglycan, ABAKAN, in the barrel field of the rat somatosensory cortex. At birth there is very little ABAKAN present in the somatosensory cortex. The levels of this proteoglycan increase during development until the highest levels are reached in the adult rat. Immunohistochemistry reveals that the TED 15 immunoreaction product marks the boundaries between cortical barrels. At postnatal day 7 (P7) the proteoglycan is localized specifically to the boundary regions. As the brain matures, the levels of ABAKAN increase in the barrel hollows and in the surrounding cortex; however the boundary regions maintain a higher level of expression even in the adult animal. The high levels of ABAKAN observed in the adult barrel fields indicate that unlike other boundary molecules this proteoglycan may be involved in maintaining the structure of the adult somatosensory cortex.

Developmental expression of KG-CAM in the rat neostriatum.

The present study examines the developmentally regulated expression pattern of an Ig superfamily member, KG-CAM, in the neostriatum of the rat. KG-CAM is a 90-kDa glycoprotein that is related to the DM-GRASP/Neurolin family of adhesion molecules. In the embryonic and early postnatal neostriatum, the distribution of KG-CAM correlates with the distribution of dopaminergic terminals. Early in neostriatal development, KG-CAM is found in the tyrosine hydroxylase-positive patches. In the maturing neostriatum, the levels of KG-CAM remain high within the patches, and KG-CAM upregulates in the matrix compartment. As the neostriatum is reaching its adult morphology, 5 weeks postnatal, the expression of KG-CAM in the matrix is approximately equal to that of the patches. When the distribution of KG-CAM is examined at the ultrastructural level, the immunoreactivity is localized to the external surface of neuronal and glial profiles in the neuropil. KG-CAM does not appear to be associated with the guidance of dopaminergic axons from the substantia nigra to the striatum, for this pathway is not immunopositive for this member of the Ig superfamily. The present study identifies an Ig superfamily member, KG-CAM, that appears to play a major role in the development of the neostriatum. Furthermore, the high levels of KG-CAM in the adult neostriatum suggest that this Ig superfamily member may be involved in maintaining the integrity of this structure in the adult rat.

Transfecting neurons and glia in the rat using pH-sensitive immunoliposomes.

Immunoliposomes were constructed using antibody 5-113 (directed to an antigen on the external surface rat glial cells), the antibody Thy 1.1, and a non-immune antibody. The antibodies were conjugated to N-gluytaryl-phosphatidylethanolamine. Liposomes were constructed with these conjugated antibodies, other lipids and a beta-galactosidase plasmid under the control of the cytomegalovirus promoter. When immunoliposomes decorated with one of three different antibodies were injected into the brain or spinal cord of adult rats, the X-gal reaction product was observed in neurons, astrocytes and vascular elements. There was an increase in neuronal labeling when animals were injected with Thy 1.1 conjugated liposomes and there was an increase in glial labeling in animals injected with 5-113 liposomes. In spinal cords, the immunoliposomes appear to penetrate a substantial distance, transfecting neurons several centimeters from the site of delivery. These data suggest that immunoliposomes may provide an effective transfection system for gene delivery in the CNS.

Delivery of plasmid DNA to glial cells using pH-sensitive immunoliposomes.

Immunoliposomes were constructed with an antibody specific to glial cells. They were used to examine the specificity and efficacy of cell type plasmid transfection. Liposomes contained a beta-galactosidase gene under control of an SV-40 promotor. Two different monoclonal antibodies of a different subclass, IgM and IgG, were examined for their targeting ability using immunoliposomes. Cultured C6 glioma (specific target cell type) and NIH 3T3 (control cell type, fibroblast) cells were transfected using these immunoliposomes. Results indicate a three-fold increase in transfection by the glial specific immunoliposomes, "gliasomes", in glial cell culture over control liposomes. Gliasomes were exposed to NIH 3T3 cells and showed no enhanced transfection over control liposomes. Gliasomes were tested for their specificity by the addition of excess antibody to the cell culture in order to saturate specific receptors on C6 glioma cells. Results indicate a reduced transfection, nearly three-fold, in cells that were saturated with excess antibody prior to exposure to the immunoliposomes.

Distribution and characteristics of a 90 kDa protein, KG-CAM, in the rat CNS.

The distribution of a 90 kDa protein, termed KG-CAM, was examined in the developing and adult rat central nervous system (CNS) using the monoclonal antibody 11-59. The amino acid sequence of this protein revealed a sequence homology with a group of chick cell adhesion molecules from the immunoglobulin superfamily: DM-GRASP; SC1; and BEN. Immunolabeling of cells cultured from the embryonic and neonatal rat brain demonstrates that the protein recognized by 11-59 is on the external surface of a subpopulation of neurons and a limited population of glial cells. When the 11-59 antibody was used to stain sections of the adult brain and spinal cord, a number of different structures were labeled. The most intense immunoreactivity was found in the somatosensory system, the basal ganglia, the cortex, the olfactory system, and the circumventricular organs. One of the more interesting aspects of KG-CAM is the spatially and temporally regulated patterns of expression observed during the development of the CNS. For example, the dendrites of layer II pyramidal cells in the granular retrosplenial cortex are immunopositive for 11-59 while the dendrites are in the process of bundling in layer I, but not before bundling begins or after the process is completed. These findings reveal the varied roles of this adhesion molecule in the developing brain and spinal cord, as well as its potential role in the maintenance of the structural integrity of the adult CNS.

Expression of a keratin sulfate proteoglycan during development of the dorsal lateral geniculate nucleus in the ferret.

ABAKAN is a keratin sulfate proteoglycan that was identified in rat brain by monoclonal antibody TED15 (Geisert et al. [1992] Brain Res. 571:165-168). It blocks neuronal attachment and neurite outgrowth in culture, is associated with astrocytes, and marks the boundaries of areas in the developing rat brain (Geisert and Bidanset [1993] Dev. Brain Res., 75:163-173). In the present study TED15 was used to examine the distribution of ABAKAN during laminar development of the dorsal lateral geniculate nucleus in ferrets. This distribution was also compared with that of astrocytes as displayed with antibodies to GFAP. In the adult, TED15 and anti-glial fibrillary acidic protein (GFAP) labeling are similar. Both are fairly uniform in the nucleus although somewhat elevated near the optic tract and in the interlaminar zone between laminae A and A1. During development the pattern is quite different. At postnatal day 1 (P1), before lamination is evident, TED15 and anti-GFAP labeling are light in the nucleus. By P10, when laminae are emerging, both are elevated in the A-A1 interlaminar zone and in the C laminae. At P18, when laminae are distinct, TED15 labels the A-A1 interlaminar zone, and it marks the borders between the ON and OFF leaflets within A and A1 (Stryker and Zahs [1983] J. Neurosci. 3:1943-1951). In comparison, anti-GFAP marks the interlaminar zone but not the ON/OFF leaflets. By 6 weeks the nucleus resembles the adult nucleus. These results show that ABAKAN marks the boundaries of the major functional subdivisions of the lateral geniculate nucleus in the developing ferret and suggest that it plays a role in lamination.

Developmental regulation of a glycolipid epitope on actively extending growth cones and central and peripheral projections in the medicinal leech.

The monoclonal antibody, A2B5, that recognizes vertebrate gangliosides also recognizes embryonic cells in the medicinal leech (Hirudo medicinalis) in a spatially and temporally regulated way. Furthermore, A2B5-positive glycolipids could be isolated from embryonic leeches. Early in development A2B5 labeled axon tracts within the central nervous system coincident with the initial growth of axons, while later in embryogenesis, A2B5 also labeled the forming peripheral nerves. This central and peripheral staining disappeared during the latter third of embryogenesis. A2B5 also labeled the growing tips of an identified myo-organizing cell. This cell, the C-cell, projects an array of parallel processes and exhibits a discrete transition in the rate of growth cone extension (J. Jellies, and W. B. Kristan, Jr. 1991. Dev. Biol. 148, 334-354.). A2B5 failed to recognize the relatively non motile growth cones of the C-cell during early embryogenesis. The C-cell growth cones began to exhibit A2B5 labeling as they became more rapidly extending and this labeling persisted throughout the later motile phase of C-cell growth. In addition to its widespread distribution on embryonic (but not mature) cellular processes, A2B5 also labeled the mitotic profiles of dividing cells in all tissues. Thus, the A2B5 epitope may be presented intracellularly, or both intra- and extracellularly. When glycolipids were extracted from embryonic leeches, partitioned by elution from a silicic acid column, and analyzed using high-performance thin-layer chromatography, at least one of the major glycolipid bands was resorcinol-positive, consistent with presentation of sialic acid residues. The fraction enriched for the resorcinol-positive band was recognized by A2B5 on dot blots, as was ganglioside GQ1b. While potential mechanisms remain to be examined, on the basis of the distinct distribution of the A2B5 epitope and our finding of A2B5-positive glycolipids in leech embryos, we suggest that complex polar glycolipids may play a role in the extension of cellular projections in the medicinal leech.

A central nervous system keratan sulfate proteoglycan: localization to boundaries in the neonatal rat brain.

During the development of the central nervous system (CNS), adhesive molecules promote the formation of axonal pathways and appropriate neuronal connections by facilitating cellular interactions. In addition to the interactions that bring neurons together, recent evidence suggests inhibition of neuronal interactions also plays a role by restricting axons to their appropriate pathways and forming boundaries between functional units of the developing CNS. The present study describes the distribution of a recently identified large keratan sulfate proteoglycan, ABAKAN, in the postnatal day 14 (P14) and adult rat brain. In the adult brain ABAKAN appears to be relatively evenly distributed throughout the CNS, while at P14 this proteoglycan is found at high concentrations between different functional units of the neonatal brain. For example, ABAKAN appears to separate different cortical areas and mark the boundaries between thalamic nuclei. In vitro assays demonstrate that this keratan sulfate proteoglycan is a potent inhibitor of neurite growth. The distribution of ABAKAN at P14 and the effects of this keratan sulfate proteoglycan on neurite growth suggest that ABAKAN functions as a molecular barrier to axonal growth in the developing rat brain.